Recombinant vaccines have been developed with the progress of recombinant DNA technology, allowing the modification of viral genomes to produce modified viruses. In this manner, it has been possible to introduce genetic sequences into non-pathogenic viruses, so that they encode immunogenic proteins to be expressed in target cells upon infection, in order to develop a specific immune response in their host.
Such vaccines constitute a major advance in vaccine technology (Kutzler et al., Nat Rev Genet, 9(10): 776-788, 2008). In particular, they have the advantage over traditional vaccines of avoiding live (attenuated) virus and eliminating risks associated with the manufacture of inactivated vaccines.
Gene delivery using modified retroviruses (retroviral vectors) was introduced in the early 1980s by Mann et al. (Cell, 33(1):153-9, 1983). The most commonly used oncogenic retroviral vectors are based on the Moloney murine leukemia virus (MLV). They have a simple genome from which the polyproteins Gag, Pol and Env are produced and are required in trans for viral replication (Breckpot et al., 2007, Gene Ther, 14(11):847-62; He et al. 2007, Expert Rev vaccines, 6(6):913-24). Sequences generally required in cis are the long terminal repeats (LTRs) and its vicinity: the inverted repeats (IR or att sites) required for integration, the packaging sequence ψ, the transport RNA-binding site (primer binding site, PBS), and some additional sequences involved in reverse transcription (the repeat R within the LTRs, and the polypurine tracts, PPT, necessary for plus strand initiation). To generate replication-defective retroviral vectors, the gag, pol, and env genes are generally entirely deleted and replaced with an expression cassette.
Retroviral vectors deriving from lentivirus genomes (i.e. lentiviral vectors) have emerged as promising tools for both gene therapy and immunotherapy purposes, because they exhibit several advantages over other viral systems. In particular, lentiviral vectors themselves are not toxic and, unlike other retroviruses, lentiviruses are capable of transducing non-dividing cells, in particular dendritic cells (He et al. 2007, Expert Rev vaccines, 6(6):913-24), allowing antigen presentation through the endogenous pathway.
Lentiviruses represent a genus of slow viruses of the Retroviridae family, which includes the human immunodeficiency viruses (HIV), the simian immunodeficiency virus (SIV), the equine infectious encephalitis virus (EIAV), the caprine arthritis encephalitis virus (CAEV), the bovine immunodeficiency virus (BIV) and the feline immunodeficiency virus (FIV). Lentiviruses can persist indefinitely in their hosts and replicate continuously at variable rates during the course of the lifelong infection. Persistent replication of the viruses in their hosts depends on their ability to circumvent host defenses.
The design of recombinant lentiviral vectors is based on the separation of the cis- and trans-acting sequences of the lentivirus. Efficient integration and replication in non-dividing cells requires the presence of two cis-acting sequences in the lentiviral genome, the central polypurine tract (cPPT) and the central terminal sequence (CTS). These lead to the formation of a triple-stranded DNA structure called the central DNA “flap”, which maximizes the efficiency of gene import into the nuclei of non-dividing cells, including dendritic cells (DCs) (Zennou et al., 2000, Cell, 101(2) 173-85; Arhel et al., 2007, EMBO J, 26(12):3025-37).
HIV-1 lentiviral vectors have been generated based on providing the subtype B Gag, Pol, Tat and Rev proteins for packaging vectors in trans from a packaging construct (Naldini et al, PNAS 15: 11382-8 (1996); Zufferey et al, Nature Biotechnology 15:871-875, 1997); Dull et al, Journal of Virology (1997)). These studies were performed with subtype B Gag and Pol proteins. The effect of non-subtype B gag and pol sequences in a HIV-1 packaging construct was not assessed.
There are many different subtypes of HIV-1 other than subtype B. Some subtypes of HIV-1, such as C, E, and A, appear to be transmitted more efficiently than HIV-1 subtype B, which is the major subtype in the United States and Europe. Essex et al., Adv Virus Res. 1999; 53:71-88. The predominant subtype of HIV-1 that is found in the developed Western World, clade B, differs considerably from those subtypes and recombinants that exist in Africa and Asia, where the vast majority of HIV-infected persons reside. Spira et al., J. Antimicrobial Chemotherapy (2003) 51, 229-240. Thus, serious discrepancies may exist between the subtype B retrovirus encountered in North America and Europe and those viral subtypes that plague humanity on a global scale. Id. Subtype diversity may impact on modes of HIV transmission. Homosexual and intravenous drug abuse are the primary modes of transmission observed for clade B strains in Europe and the Americas. Id. In contrast, clades A, C, D and E predominate in Africa and Asia where heterosexual transmission predominates. Id. In addition, some studies suggest that AIDS progression differs as a function of infecting subtype. Id. Thus, it appears that HIV-1 subtype B is quite different than the other HIV-1 subtypes.
HIV phylogenic classifications are normally based either on nucleotide sequences derived from multiple sub genomic regions (gag, pol and env) of the same isolates, or on full-length genome sequence analysis. A phylogenic analysis of HIV-1 near-full length sequences revealed that HIV-1 subtype B was most closely related genetically to HIV subtype D (FIG. 1). Phylogenic analyses of HIV-1 Gag and Pol protein sequences also showed that HIV-1 subtype B was most closely related genetically to HIV subtype D (FIG. 2).
Nevertheless, HIV-1-NDK, a subtype D virus, is significantly more cytopathic for CD4+ lymphocytes than the HIV-1-BRU prototype, a subtype B virus. This may be due to enhanced fusogenicity and infectivity of subtype D viruses. De Mareuil et al., J. Virol. 66: 6797 (1992). Phenotypic analysis of recombinant viruses indicated that 75 amino acids from the N-terminal part of HIV-1-NDK matrix (MA) protein, together with the HIV-1-NDK envelope glycoprotein, are responsible for enhanced fusogenicity of HIV-1-NDK in CD4+ lymphocytes as well as for enhanced infectivity of HIV-1-NDK in some CD4-cell lines. Id.
There is a need in the art for lentiviral packaging constructs producing higher titers of packaged lentiviral vectors, in order to reduce injection volumes, increase dosages, reduce the cost of vaccination, and increase the number of patients that could be treated with one batch. The current invention fulfills this need.